Magnetic separation using nanoparticles

ABSTRACT

Nanoparticles as described herein are configured to bind to bacterial contaminants, such as Gram positive bacteria, Gram negative bacteria, and endotoxins. The nanoparticles include a core comprising a magnetic material; and a plurality of ligands attached to the core. The ligands include, for example, bis(dipicolylamine) (“DPA”) coordinated with a metal ion, e.g., Zn 2+  or Cu 2+ , to form, e.g., bis-Zn-DPA or bis-Cu-DPA, which can bind to the bacterial contaminants. The nanoparticles can be included in compositions for use in methods and systems to separate bacterial contaminants from liquids, such as liquids, such as blood, e.g., whole or diluted blood, buffer solutions, albumin solutions, beverages for human and/or animal consumption, e.g., drinking water, liquid medications for humans and/or animals, or other liquids.

CLAIM OF PRIORITY

This application claims priority to U.S. Provisional Application Ser.No. 61/758,046, filed on Jan. 29, 2013, the entire contents of which arehereby incorporated by reference.

FIELD OF THE INVENTION

The invention relates to magnetic separation, e.g., of bacteria andendotoxins, using nanoparticles.

BACKGROUND OF THE INVENTION

Bacterial sepsis is a serious clinical condition that can lead tomultiple organ dysfunction and death despite timely treatment withantibiotics and fluid resuscitation. Gram-negative bacteria are commoncausative pathogens in bacterial sepsis. Common treatments for sepsisand septic shock include antibiotics, fluid resuscitation, andvasoactive medications. Other approaches, such as extracorporeal removalof inflammatory mediators, corticosteroids, and recombinant protein drugtherapies have been introduced as experimental adjunct treatments.Antibody-based methods of separating bacteria from blood have also beendeveloped.

SUMMARY OF THE INVENTION

The invention is based, at least in part, on the discovery thatnanoparticles, such as magnetic nanoparticles, modified with ligandsincluding metal ion-coordinated bis(dipicolylamine) (e.g., zincion-coordinated bis-Zn-DPA or copper ion-coordinatedbis-Cu-DPA)(collectively, bis-M-DPA), can be utilized for highlyselective and rapid separation of bacterial contaminants, such asbacteria, e.g., E. coli, and endotoxin, from liquids, such as blood,e.g., whole or diluted blood or serum, buffer solutions, albuminsolutions, beverages for human and/or animal consumption, e.g., drinkingwater, liquid medications for humans and/or animals, or other liquids.

When bis-M-DPA modified nanoparticles are mixed with the liquid, e.g.,blood, rapid binding occurs between the bis-M-DPA ligands on one or morenanoparticles and bacterial contaminants in the liquid. A motive force,e.g., a magnetic force, or other means of separation, e.g., size-basedseparation, centrifugal force separation, or flow cytometry-basedseparation, can then be applied to the nanoparticles to separate themfrom the liquid. Some of the nanoparticles have bacteria or endotoxinbound thereto, and when the nanoparticles are removed, the contaminantsare also removed with them from the liquid. Microfluidic systems can beused as platforms for nanoparticle-based separations, providing amechanism for efficient and selective separation of contaminants fromliquids of any kind.

In one general aspect, nanoparticles are configured to bind to one ormore bacterial contaminants. The nanoparticles include a core comprisingor consisting of a magnetic material; and a plurality of ligandsattached to the core. The ligands include bis(dipicolylamine)coordinated with a metal ion, e.g., Zn²⁺ or Cu²⁺ (bis-M-DPA), which canbind to the bacterial contaminant.

Various embodiments of these compositions can include one or more of thefollowing features. The bis-M-DPA can be attached to the core via aspacer molecule having a molecular weight of 10 kDa to 50 kDa, e.g., 15,20, 25, 30, 35, 40, or 45 kDa. In some cases, the spacer molecule can bea polymer spacer comprising or consisting of polyethylene glycol (PEG).The ligands can further include a filler molecule attached to the core.In some cases, the filler molecule can include or consist of PEG. Thecore can include one or more of a ferrimagnetic material, aferromagnetic material, an antiferromagnetic material, a paramagneticmaterial, and a superparamagnetic material. In some cases, the coreincludes or consists of iron oxide, e.g., Fe₃O₄. An external surface ofthe core can also include or consist of a chemical group that can bindto the ligands.

In some embodiments, the diameter of the core can be about 50 to 250 nm,e.g., 60, 70, 80, 90, or 100 nm.

The nanoparticles can be configured such that binding between thenanoparticles and the bacterial contaminant can be substantiallycomplete, e.g., 90% of the nanoparticles are bound to bacteria, withinan incubation time of about five minutes or less, e.g., about one, two,three, or four minutes or less.

The bacterial contaminants can be or include Gram positive bacteria,Gram negative bacteria, and/or bacterial endotoxins.

In another general aspect, methods of separating bacterial contaminantsfrom a liquid include combining the liquid with a plurality ofnanoparticles that can bind to the bacterial contaminants. Eachnanoparticle includes a core including a magnetic material and aplurality of ligands attached to the core. The ligands includebis(dipicolylamine) coordinated with Zn²⁺ or Cu²⁺ (bis-M-DPA). Themethods further include applying a magnetic field to the liquidincluding the nanoparticles such that at least some of the nanoparticlesare attracted by the magnetic field. The methods further includeseparating the liquid from the attracted nanoparticles. At least some ofthe bacterial contaminants, if present in the liquid, are bound to atleast some of the attracted nanoparticles.

Various embodiments of these methods can include one or more of thefollowing features. The bacterial contaminants can be or include Grampositive bacteria, Gram negative bacteria, and/or bacterial endotoxins.The liquid can be or include blood. The methods can further includedirecting the liquid including the nanoparticles through a flow channel.In some cases, applying a magnetic field includes applying a magneticfield across the flow channel. In some embodiments, separating theliquid includes allowing the liquid to exit the flow channel, andwherein the attracted nanoparticles remain in the flow channel. In somecases, directing the liquid through the flow channel includes flowingthe liquid through the flow channel with a flow rate of at least about5, 6, 7, 8, 9, or 10 mL/hour, or much faster, e.g., at least 40, 50, 60,or 70 mL/hour. In some cases, the methods further include directing theliquid including the nanoparticles through a plurality of flow channels.Applying a magnetic field can include applying a magnetic field acrosseach of the plurality of flow channels.

The methods can further include incubating the liquid including thenanoparticles prior to applying the magnetic field. In some cases,incubating the liquid including the nanoparticles includes incubatingfor about five minutes or less, e.g., about one, two, three, or fourminutes or less, to achieve substantially complete binding between thenanoparticles and the bacterial contaminant.

In another general aspect, systems for separating one or more bacterialcontaminants from a liquid include a plurality of nanoparticles that canbind to the target species. Each nanoparticle includes a core comprisinga magnetic material, and a plurality of ligands attached to the core.The ligands include bis(dipicolylamine) coordinated with a metal, e.g.,Zn²⁺ or Cu²⁺ (bis-M-DPA). The systems further include a source of amagnetic field arranged to apply the magnetic field to a liquidincluding the plurality of nanoparticles. At least some of thenanoparticles are attracted by the magnetic field. The system furtherincludes a flow path for separating the liquid from the attractednanoparticles. Some of the bacterial contaminants, if present in theliquid, are bound to at least some of the attracted nanoparticles.

Different embodiments of these systems can include one or more of thefollowing features. The systems can further include a flow channelconfigured to receive the liquid including the nanoparticles. In somecases, the magnet is disposed to a side of the flow channel andconfigured to apply the magnetic field across the flow channel. In somecases, the flow path includes an exit from the flow channel, and whereinthe attracted nanoparticles remain in the flow channel. In some cases,the system further includes a plurality of flow channels configured toreceive the liquid including the nanoparticles.

The nanoparticles can be configured such that binding between thenanoparticles and the bacterial contaminants can be substantiallycomplete within an incubation time of about five minutes or less, e.g.,about one minute or less. The bacterial contaminants include Grampositive bacteria, Gram negative bacteria, and/or bacterial endotoxins.The liquid can be or include blood.

The term “bacterial contaminants” refers to bacteria and endotoxins ofany species and a liquid may contain any type of bacteria, or multipletypes of bacteria, or only endotoxins, or both bacteria and endotoxins.

The approaches described herein for bacterial contaminant removal, e.g.,magnetic separation, from fluids have several advantages. For instance,magnetic separation using nanoparticles is generic and can be used onmany target bacterial contaminant species, including Gram positivebacteria, Gram negative bacteria, and/or bacterial endotoxins.Separation using nanoparticles is also highly selective; thenanoparticles have negligible affinity toward normal mammalian cells andother materials that may be present in the fluids. Binding betweennanoparticles and a target species is rapid and very short incubationtimes can be used. In addition, separation can be achieved at rapid flowrates through microfluidic systems, enabling efficient separation ofbacterial contaminants from fluids. The synthetic ligands used areinexpensive and straightforward to synthesize, can be easily conjugatedto nanoparticles, and are stable against denaturation at roomtemperature and even at body temperature.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentspecification, including definitions, will control. In addition, thematerials, methods, and examples are illustrative only and not intendedto be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of a nanoparticle binding to a bacterial cell.

FIG. 2A is a diagram of a nanoparticle.

FIG. 2B is a diagram of an active ligand molecule.

FIG. 3 is a flow chart of a process for making nanoparticles.

FIG. 4 is a flow chart of a process for magnetic separation.

FIG. 5 is a diagram of a dual-inlet microfluidic system.

FIG. 6 is a diagram of a single-inlet microfluidic system.

FIG. 7 is a diagram of a networked microfluidic system.

FIGS. 8A and 8B are schemes for the modification of magneticnanoparticles with ligand molecules.

FIGS. 9A-9C are energy dispersive X-ray spectra for nanoparticles ofvarious compositions mixed with a zinc compound.

FIG. 10A-10F are phase contrast, green fluorescent, and overlay imagesof nanoparticles and E. coli.

FIG. 11 is a plot of the concentration of E. coli after magneticseparation.

FIG. 12 is a plot showing a correlation between fluorescent labelinganalysis and colony counting analysis.

FIG. 13 is a plot of the concentration of endotoxin after magneticseparation.

FIGS. 14A and 14B are plots of the concentration of E. coli and redblood cells in whole blood after magnetic separation.

FIGS. 15A-15E are phase contrast and green fluorescent images ofnanoparticles and E. coli in a dual inlet microfluidic system.

FIG. 16 is a plot of the concentration of E. coli after passing througha dual inlet microfluidic system.

FIGS. 17A-17F are phase contrast, green fluorescent, and overlay imagesof nanoparticles and E. coli in a single inlet microfluidic system.

FIG. 18 is a plot of the concentration of E. coli after passing througha single inlet microfluidic system.

FIG. 19A-19D are a series of plots of the concentration of E. coli afterpassing through a network of microfluidic systems at various flow rates.

FIG. 20 is a scheme for the synthesis of bis-DPA.

FIG. 21 is a scheme for the synthesis of bis-DPA-PEG-COOH.

FIG. 22 is a scheme for the synthesis of bis-Zn-DPA-PEG-FITC.

DETAILED DESCRIPTION

Referring to FIG. 1, nanoparticles, e.g., magnetic nanoparticles 100modified with ligands 102 including metal-coordinatedbis(dipicolylamine) (e.g., bis-Zn-DPA or bis-Cu-DPA) (referred tocollectively herein as “bis-M-DPA”) can be utilized for highly selectiveand rapid separation of bacterial contaminants such as bacteria 104,e.g., E. coli, and endotoxins, from liquids such as whole blood,buffers, albumin, and other liquids. When nanoparticles 100 are mixedwith blood, rapid binding (e.g., within about one minute or less) occursbetween the bis-M-DPA ligands on one or more nanoparticles 100 and thetarget bacteria 104 or endotoxin, forming nanoparticle-contaminantcomplexes 105. If the nanoparticles are magnetic or include a magneticmaterial, a magnetic field applied to the blood attracts nanoparticles100, some of which have bacteria or endotoxin bound thereto, thusreducing the concentration of bacterial contaminants in the blood.Stationary magnetic separation with a magnet 106 can be used to separatebacterial contaminants bound to nanoparticles from the liquid.Microfluidic systems can also be used as a platform fornanoparticle-based magnetic separations.

In other implementations, the nanoparticles bound to the bacterialcontaminants are designed to be a size that of can be separated from thefluid using a microfluidic system, e.g., as described in U.S. Pat. No.8,304,230, which separates the particles based on size. Alternatively,the nanoparticles bound to the bacterial contaminants can be removedfrom the fluid using centrifugation or sedimentation. In otherimplementations, microparticles can be used to bind bacterialcontaminants.

Nanoparticle Compositions and Synthesis

Referring to FIG. 2A, magnetic nanoparticles 200 include a magnetic core202 and ligand molecules 204. In general, the core is formed of amagnetically susceptible material. For instance, the core 202 can be aferrimagnetic material such as, e.g., iron oxide (Fe₃O₄), or anotherferrimagnetic material. The core 202 can also be an antiferromagneticmaterial, such as, e.g., chromium oxide (Cr₂O₃). The core 202 can alsobe a paramagnetic material, such as, e.g., lanthanide oxide, manganeseoxide, or another paramagnetic material. The core 202 can also be asuperparamagnetic material, such as, e.g., iron oxide (Fe₂O₃). The core202 can also be a ferromagnetic material, such as, e.g., nickel, cobalt,alnico (an aluminum-nickel-cobalt alloy of iron, or anotherferromagnetic material. In some implementations, the nanoparticles canbe cross-linked iron nanoparticles or monocrystalline ironnanoparticles, such as those described in U.S. Patent Pub. 2009/0029392,the contents of which are incorporated herein by reference. In someexamples, the outer surface of the core 202 can include chemical groupsthat are capable of binding to the ligand molecules 204. For instance,the outer surface of the core 202 can include carboxylic acids, amines,thiols, bromoacetyl compounds, alcohols, or another chemical group.

The ligand molecules 204 include active ligand molecules 206 that arecapable of binding to the target bacterial contaminant species, such asGram-positive bacteria, Gram-negative bacteria, and endotoxins. Forinstance, the active ligand molecules can include zinc-coordinatedbis(dipicolylamine) (bis-Zn-DPA) 208, copper-coordinated DPA(bis-Cu-DPA), other another molecule. Bis-M-DPA forms coordination bondswith anionic phospholipids, which are present at high density on theouter membrane of Gram-positive and Gram-negative bacterial cells.Furthermore, the bis-M-DPA coordination complex with anionicphospholipids provides negligible affinity toward normal mammalian cellssuch as red blood cells or white blood cells or toward negativelycharged proteins such as albumin. Bis-M-DPA is thus capable of highlyselective binding to Gram-positive bacteria, Gram-negative bacteria, andendotoxin with little to no binding to normal cells and proteins.

Referring also to FIG. 2B, in one example of an active ligand module206, bis-Zn-DPA can be attached to the nanoparticles 200 via a spacergroup 210. The spacer group 210 provides space between the core 202 andbis-Zn-DPA 208 to avoid crowding among the bulky bis-Zn-DPA groups andto enhance the molecular mobility of the bis-Zn-DPA groups. The spacergroup 210 can be a polymer, such as polyethylene, polyethylene glycol(PEG), polypropylene, or another polymer. In one example, PEG with amolecular weight (MW) of about 3.5, 5.0, 10, 15, 20, 25, 40, or 50 kDacan be used as the spacer group 210. The spacer group 210 is terminatedwith an end group capable of binding to the core 202 or to the chemicalgroups 206 on the outer surface of the core 202. For instance, the endgroup can be, e.g., a carboxylic acid (e.g., capable of binding to asurface amine via carbodiimide chemistry), an amine (e.g., capable ofbinding to a surface carboxylic acid via carbodiimide chemistry), athiol (e.g., capable of binding to a surface thiol via a disulfide bondor via reaction with a maleimide), a bromoacetyl compound (e.g., capableof binding to a surface via a nucleophilic addition), alcohol (capableof binding to a surface via silane chemistry), or another end group. Insome examples, there can be about 2,500 active ligand molecules 206 per100 nm diameter nanoparticle 200, e.g., as characterized in (Sofia, S.J., Premnath, V., Merill, E. W., 1998. Poly(ethylene oxide) grafted tosilicon: grafting density and protein adsorption. Macromolecules 31,5059-5070), the contents of which are incorporated herein by reference.

The ligand molecules 204 can also include filler molecules 212 thatpassivate binding sites on the core 202 (e.g., chemical groups on theouter surface of the core) to which a bis-Zn-DPA ligand is not bound,thus preventing non-specific binding to those unreacted binding sites.The filler molecules 212 can be polymers, such as, e.g., polyethylene,PEG, polypropylene, or another polymer. Each filler molecule 212 isterminated with an end group capable of binding to the core 202 (e.g.,to the chemical groups on the surface of the core). For instance, theend group can be, e.g., a carboxylic acid, a thiol, an amine, or anotherend group.

In one example, a Fe₃O₄ core 202 includes amines on its outer surface.The active ligand molecules 206 are formed of bis-Zn-DPA with acarboxylic acid-terminated PEG spacer. This example of a ligandcomposition is referred to herein as bis-DPA-PEG-COOH. The fillermolecules 212 are carboxylic acid-terminated PEG. Bis-DPA-PEG-COOH iscapable of rapid binding to Gram positive and Gram negative bacteria andto endotoxin. In addition, bis-DPA-PEG-COOH is stable at roomtemperature and thus has a long shelf life, and is also stable at bodytemperature and thus well suited to use in clinical applications.

The size of the core 202 is generally less than about severalmicrometers, e.g., about 100 nm or less. In some examples, the minimumcore size may be limited by the size of the magnetic moment of thenanoparticle, e.g., depending on the strength of the magnet used formagnetic separation. A smaller core provides a larger surface area tovolume ratio and thus can provide better performance in clinicalapplications (e.g., more rapid binding to the target species). In someexamples, reducing the size of the core 202 below a threshold size canreduce the magnetic moment of the core, thus reducing the ability of thenanoparticles to respond to a magnetic field. For instance, for Fe₃O₄nanoparticles, the magnetic moment of the core begins to decrease forcore sizes less than about 50 nm.

FIG. 3 is a flowchart showing a process for synthesizing thenanoparticles of FIG. 2. Ligand molecules including bis-DPA (referred toherein as “bis-DPA ligands”) are synthesized (300) by synthesizing3-(3,5-bis((bis(pyridin-2-ylmethyl)amino)methyl)-4-hydroxyphenyl)propanoic acid (bis-DPA) (302) and attaching a spacer group (304). Forinstance, bis-DPA can be synthesized by a solution-phase preparation ofmethyl3-(3,5-bis((bis(pyridin-2-ylmethyl)amino)methyl)-4-hydroxyphenyl)propanoate,bis-DPA-methyl ester (bis-DPA-ME) and the conversion of bis-DPA-ME intobis-DPA. A spacer group (e.g., PEG) terminated with an end group (e.g.,COOH) can be attached to bis-DPA through any of a variety of chemicalreactions. For instance, carbodiimide chemistry can be used to formbis-DPA-PEG-COOH through the reaction of bis-DPA and NH2-PEG-COOH in thepresence of 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride(EDC) and N-hydroxysuccinimide (NHS).

Bis-DPA ligands (e.g., PEG-DPA) are attached to the nanoparticle cores(306) via any number of standard chemical reactions between the endgroups of the ligands (e.g., COOH) and the chemical groups on the outersurface of the nanoparticle cores using standard techniques. Forinstance, carbodiimide chemistry can be used to attach PEG-DPA toamine-terminated Fe₃O₄ nanoparticles through the reaction of PEG-DPA andthe nanoparticles in the presence of EDC and NHS. Other chemistries canalso be used, e.g., based on the identity of the ligand end groups andthe chemical groups on the surface of the nanoparticles.

Unreacted chemical groups on the surface of the nanoparticles arepassivated (308) with filler molecules. For instance, PEG-succinimidylvalerate (PEG-SVA) can be used to passivate the nanoparticle surface byreacting PEG-SVA with amine-terminated Fe₃O₄ nanoparticles. Otherchemistries can also be used, e.g., based on the chemical groups on thesurface of the nanoparticles.

Zinc (e.g., Zn²⁺) is coordinated to the bis-DPA groups on thenanoparticles (310) to create bis-Zn-DPA-PEG modified nanoparticles(referred to herein as NP_(PEG-DPA-Zn)). For instance, zinc can becoordinated to bis-DPA by stirring a solution of nanoparticles in thepresence of a zinc compound, such as zinc nitrate hexahydrate. Inanother example, copper (e.g., Cu²⁺) can be coordinated to the bis-DPAgroups on the nanoparticles to create bis-Cu-DPA-PEG modifiednanoparticles. In another example, bis-Cu-DPA-PEG can be directlyattached to the nanoparticle cores.

The resulting nanoparticles include active ligands including, e.g.,bis-Zn-DPA (e.g., bis-Zn-DPA-PEG) or bis-Cu-DPA (e.g., bis-Cu-DPA-PEG)capable of binding to target species, such as Gram-positive bacteria,Gram-negative bacteria, and endotoxins; and filler molecules (e.g.,PEG-SVA). These nanoparticles are referred to herein as NP_(DPA-Zn)(e.g., NP_(PEG-DPA-Zn)) or NP_(DPA-Cu), or collectively as NP_(DPA-M).

Magnetic Separation of Bacterial Contaminants

Referring to FIG. 4, magnetophoresis can be used to clear bacterialcontaminants such as Gram-positive bacteria, Gram-negative bacteria, andendotoxins, from liquids such as blood using NP_(DPA-M). Referring alsoto FIG. 1, for example, NP_(DPA-M) 100 are combined (400) with acontaminated sample of blood 108 and incubated (402) to allow bindingbetween bis-M-DPA and contaminants 104 in the blood 108. In someexamples, multiple nanoparticles 100 bind to a single contaminant 104(e.g., a single bacterial cell). The concentration of nanoparticles inthe blood can be from about 1.0×10⁴/mL to about 1.0×10¹²/mL, e.g., about1.0×10¹¹/mL.

The binding kinetics between bis-M-DPA on nanoparticles and bloodcontaminants are rapid. For instance, incubation times less than fiveminutes (e.g., less than one minute) can be sufficient for binding to besubstantially complete, e.g., such that a further increase in incubationtime may not cause a further increase in binding. Without being bound bytheory, it is believed that this rapid binding rate can be attributableto the high mobility of nanoparticles, which can increase theprobability of an encounter between a nanoparticle and a contaminant;and/or the high surface area to volume ratio of nanoparticles, which canincrease the ligand loading capacity of the nanoparticles. The rapidbinding rate can also be attributed to the presence of spacer groups ofthe appropriate length (e.g., between about 1.0 and 20 kDa), such as PEGspacers, that enhance the molecular mobility of the bis-M-DPA groups onthe ligand molecules and the surface of the nanoparticles.

After incubation, a magnetic field is applied (404) to the mixture ofblood 108 and nanoparticles 100. For instance, a magnet 106 or anelectromagnet can be used to apply a magnetic field to the mixture. Themagnetic nanoparticles 100 move relative to the blood 108. For instance,the nanoparticles 100 can move toward the magnet 106. Contaminants 104that are bound to the nanoparticles 100 are also moved along with thenanoparticles 100, e.g., toward the magnet 106. In the example depictedin FIG. 5B, one nanoparticle 100 is shown binding to each contaminant104. In some examples, multiple nanoparticles 100 can bind to eachcontaminant 104.

The attracted nanoparticles 100, some of which have contaminants 104bound thereto, are separated (406) from the blood 108. For instance, inthe example of FIG. 1, a stationary magnetic separation is carried out,creating an accumulation 110 of nanoparticles, from which decontaminateblood 114 can be decanted, e.g., with a syringe 112. In other examples,magnetic separation can occur in a flow cell such that the blood 108flows past the separated nanoparticles 100 and exits the flow cell.

In some examples, the magnetic separation process is repeated (408),using fresh nanoparticles for each iteration of the process. Forinstance, the magnetic separation process can be repeated a fixed numberof times (e.g., two times, three times, or another number of times). Themagnetic separation process can also be repeated based on the results ofa test, e.g., indicative of a level of contaminants in the blood. Forinstance, the magnetic separation process can be repeated until thelevel of contaminants falls below a threshold level (410).

In some examples, centrifugation can be used to remove nanoparticlesbound to contaminants.

Microfluidic Systems for Magnetic Separation

Magnetic separation of contaminants from blood using NP_(DPA-M) can beachieved with microfluidic systems. Microfluidic technologies are oftenwell-suited to the separation and sorting of cells under continuousfluid flow. In a system for microfluidic magnetic separation ofcontaminants, a mixture of blood and NP_(DPA-M) is injected into aninlet of a microfluidic system. A magnet, e.g., a permanent magnet orelectromagnet, along the flow path through the system applies a magneticfield to the blood within the system, attracting the nanoparticles withbound contaminants and preventing them from flowing to the outlet of thesystem with the blood.

In other implementations, other forces and techniques can be used toremove the nanoparticles bound to bacterial contaminants from the liquidsample. For example, the nanoparticles can be bound to reporter groups,e.g., fluorescent molecules, which can be used to selectively separatethe marked nanoparticles from the fluid, e.g., using flow cytometry oroptical forces using standard techniques. Other methods based onsize-separation techniques can also be used. For example, thenanoparticles can be bound to particles of a certain size and thenpassed through a microfluidic size-separation device known in the field.

Referring to FIG. 5, in one implementation, a dual inlet microfluidicsystem 500 includes two inlets (inlet A and inlet B) and two outlets(outlet C and outlet D). One or more magnets 510 are positioned along acommon flow channel 512 between the inlets A and B and the outlets C andD to apply a magnetic field to the liquid within the system 500.

In one example, saline is injected into inlet A and a liquid mixture ofNP_(DPA-M) and contaminants such as E. coli (e.g., as whole blood or ina buffer such as phosphate buffered saline (PBS) is injected into inletB. In the absence of a magnetic field and at a sufficiently low flowrate (e.g., about 9.0 mL/hour or less), convective mixing between twointerfacing parallel flows was negligible and flow through the commonflow channel 512 is laminar. Thus, in the absence of a magnetic field,nanoparticles, contaminants, and any other components of the liquidmixture entering the flow channel 512 from inlet B will exit throughoutlet D. The saline solution exiting through outlet C will containlittle or no nanoparticles or contaminants. In the presence of amagnetic field, nanoparticles are drawn into the saline flow and exitthrough outlet C, bringing along any contaminants bound thereto. Theliquid exiting through outlet D is thus relatively more free ofcontaminants than was the liquid entering into inlet B. In someimplementations, the liquid can be run through the system again tofurther reduce the level of bacterial contaminants.

Referring to FIG. 6, in another implementation, a single inletmicrofluidic system 600 includes a single inlet (inlet B) and twooutlets (outlet C and outlet D). One or more magnets 610 are positionedalong a common flow channel 612 between inlet B and outlets C and D toapply a magnetic field to the liquid within the system 600. A liquidmixture of NP_(DPA-M) and contaminants (e.g., as whole blood or in abuffer such as PBS) is injected into inlet B. Separation of contaminantsfrom blood is effected by the accumulation of NP_(DPA-M) at the walls ofthe common flow channel 612 adjacent to the magnets 610, instead of byremoval through one of the two outlets. The liquid exiting through bothoutlet C and outlet D is thus relatively more free of contaminants thanwas the liquid entering into inlet B.

A system of nested or multiplexed microfluidic devices can enhance theseparation efficiency by allowing multiple magnetic separations to becompleted in a single pass through the system. Individual single inletmicrofluidic systems (e.g., such as system 600) can be connected invarious combinations, such as in series, in parallel, or both, toprovide a desired number of magnetic separations per pass through thesystem.

Referring to FIG. 7, in another implementation, a nested system 700includes four individual single inlet microfluidic systems 600. InletsA, E, and F were closed so that inlet B was the only inlet into thenested system 700. A liquid mixture of NP_(DPA-M) and contaminants(e.g., as whole blood or in a buffer such as PBS) is injected into thesingle inlet B. Liquid exits at outlets G and H after passing throughtwo magnetic separations effected by magnets 710 and thus has aconcentration of contaminants that is less than the concentration of theinjected liquid. Liquid exits at outlets I and J after passing throughthree magnetic separations and thus has a concentration of contaminantsthat is less than the concentration of contaminants in the liquid thatexits at outlets G and H. In some examples, significant reductions inthe concentration of contaminants (e.g., reductions of at least about80%, or at least about 95%) can be achieved by the example nested system700.

Magnetic separation using a microfluidic system, such as a single inletmicrofluidic system, can be achieved at high flow rates of liquidthrough the system. For example, contaminants can be separated fromliquid with a flow rate of at least about 9 mL/hour (e.g., at leastabout 18 mL/hour, at least about 30 mL/hour, or at least about 60mL/hour).

Applications for Magnetic Separation Using Nanoparticles

The short incubation time for binding contaminants to NP_(DPA-M) and theability to use high flow rates through microfluidic systems render theuse of NP_(DPA-M) to separate contaminants from liquids such as bloodmake the new methods described herein well-suited for clinicalapplications. Minimizing the time that blood spends outside the bodyduring decontamination reduces the risk of coagulation and the risk ofinfection. Furthermore, the present nanoparticle-based approach toseparating bacterial contaminants from blood is compatible with existingblood-modifying technology used in intensive care units, such aspheresis, dialysis, and Extracorporeal Membrane Oxygenation (ECMO).

Sepsis is a highly lethal condition that frequently arises from thepresence of bacteria in the bloodstream. While bacteremia is animportant component of the pathogenesis of sepsis, the release ofendotoxins from bacteria is a major causative factor in septic shock.The new methods described herein provide an efficient mechanism forclearing Gram-negative bacteria, such as E. coli, endotoxins, and otherbacterial contaminants from the bloodstream of septic patients.

To further improve the efficiency of the nanoparticle-based approachdescribed herein, the accumulation of nanoparticles near the magnets inmicrofluidic systems can be reduced. In some implementations, theaccumulation of nanoparticles near the magnets can gradually reduce theefficiency of the magnetic separation, thus limiting the volume of bloodthat could be filtered before cleaning or replacing the microfluidicsystem. This potential problem can be addressed by employing strongermagnets, using an automated flushing system, e.g., a back-flushingsystem, to remove the accumulated nanoparticles, and/or increasing thecontact area between the magnets and the flow channels to mitigatenanoparticle accumulation near the magnets. In addition, lowerconcentrations of nanoparticles can be used, and/or blood can be dilutedprior to processing.

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

The following examples generally show the synthesis and characterizationof nanoparticles modified with bis-DPA-PEG-Zn. The examples further showthe ability of these nanoparticles to magnetically separate E. coli andendotoxin from liquids and from whole blood. In addition, examples ofmicrofluidic systems that can be used to implement the magneticseparation of E. coli and endotoxin from blood are demonstrated.

Example 1 Nanoparticle Synthesis and Characterization

FIG. 8A shows a scheme 800 for the preparation of magnetic nanoparticlesmodified with bis-DPA-PEG-Zn (referred to herein as NP_(PEG-DPA-Zn)).Bis-DPA with a PEG (MW=10 kDa) spacer (bis-DPA-PEG-COOH) was immobilizedon the surface of 100 nm diameter amine-terminated Fe₃O₄ magneticnanoparticles (approximately 300 amines per nanoparticle, SiMAG-Amineparticles, chemicell GmbH, Berlin, Germany) through carbodiimidechemistry using EDC and (sulfo-NHS). Excess bis-DPA-PEG-COOH wasactivated by N-hydroxysulfosuccinimide (sulfo-NHS) with EDC (10-foldmolar excess each) and added to a solution of nanoparticles. Thenanoparticle solution was washed twice with carbonate buffer (50 mMsodium carbonate, pH 9.5) using a magnetic separator (MagnetoPURE,chemicell GmbH). After 2 hours of reaction at room temperature, themodified nanoparticles were washed twice with carbonated buffer.

Unreacted free amines on the surface of the nanoparticles werepassivated with PEG-succinimidyl valerate (PEG-SVA). PEG-SVA (2.0 kDa,10-fold molar excess, Laysan Bio, Inc., Arab, Ala.) was added to thenanoparticle solution and allowed to react for two hours. Thenanoparticles were then washed twice with ddH2O to obtain nanoparticlesmodified with bis-DPA-PEG and PEG-SVA using a magnetic separator. Zn²⁺was coordinated to the bis-DPA to create bis-DPA-PEG-Zn modifiednanoparticles (NP_(PEG-DPA-Zn)). Excess of zinc nitrate hexahydrate wasadded to the NP_(PEG-DPA) solution and then the mixture was stirred forminutes at room temperature. Excess of Zn²⁺ was removed bymagnetophoresis (3 times).

FIG. 8B shows a scheme 802 for the preparation of a control sample ofmagnetic nanoparticles modified with PEG-SVA (referred to herein asNP_(PEG)). NP_(PEG) were prepared in the same manner as NP_(PEG-DPA-Zn)but without the addition of the bis-DPA-PEG-COOH.

Carbodiimide chemistry on the modified nanoparticles was validated bymeasurements of the zeta potentials of the nanoparticles. The zetapotentials of the nanoparticles were measured at room temperature with aDelsa Nano C zeta potential instrument (Beckman Coulter, Brea, Calif.).Data given below are means±standard deviations. N=4 for all groups.

Referring to Table 1, a significant decrease in zeta potential wasobserved upon addition of PEG-SVA to a solution of unmodifiednanoparticles (from 39 mV for unmodified nanoparticles to 12 mV forNP_(PEG), p<0.01). When excess PEG-SVA was added to bis-DPA-modifiednanoparticles (NP_(DPA)) to remove unreacted amines, the zeta potentialdecreased from 36 mV for NP_(DPA) to 32 mV for NP_(PEG-DPA) (p<0.01).These results validate the carbodiimide chemistry on the surface of thenanoparticles.

TABLE 1 Zeta potential (mV) of Nanoparticles NP_(amine) NP_(DPA)NP_(PEG-DPA) NP_(PEG-DPA-Zn) NP_(PEG) 39.25 ± 0.67 36.00 ± 1.36 31.70 ±1.64 33.22 ± 1.14 12.63 ± 0.96

The association of Zn with bis-DPA was demonstrated with energydispersive X-ray spectroscopy (EDS) in a scanning electron microscope(SEM). SEM imaging was performed with a JEOL 5910 microscope (JEOL,Tokyo, Japan) equipped with a detector for EDS. Prior to SEM analysis ofnanoparticles, nanoparticles were mixed with Zn(NO₃)₂, washed threetimes with deionized water to remove excess Zn(NO₃)₂, and freeze-dried.The nanoparticles aggregated during the freeze-drying process. The driednanoparticles were placed on carbon double-sided tape. Imaging wasperformed with 20 kV acceleration voltage.

EDS was performed for samples prepared from the following nanoparticlecompositions mixed with Zn(NO₃)₂: NP_(PEG-DPA), NP_(PEG), and unmodifiednanoparticles. FIGS. 9A, 9B, and 9C show EDS spectra 900, 902, 904,respectively, for each of these samples. A significant Zn signal wasdetected in the spectrum 900 the NP_(PEG-DPA) sample; the NP_(PEG) andunmodified nanoparticle spectra showed only trace amounts of Zn.Secondary electron mode SEM images and EDS images of iron and zinc onthese samples show the presence of iron in all three samples but confirmthat zinc is present only in the NP_(PEG-DPA) sample. Furthermore, theoverlap of the iron and zinc signals in the EDS images for theNP_(PEG-DPA) sample indicate the chelation of Zn²⁺ with bis-DPA in thissample. These results confirmed that both bis-Zn-DPA-PEG and PEG weresuccessfully immobilized on the surface of nanoparticles.

Example 2 Fluorescence Visualization of E. Coli Binding to Nanoparticles

Fluorescence-labeled E. coli were used to demonstrate the bindingcapability of NP_(PEG-DPA-Zn). A bacterial cell culture was prepared byinoculating 25 mL of standard lysogeny broth (LB) with E. coli (E. colistrain Stbl3, Life Technologies, Grand Island, N.Y.) using standardsterile technique. This culture was incubated for 12-18 hours at 37° C.and 5.0% CO₂ and agitated at 200 RPM in an orbital shaker incubatorovernight. Before using the bacteria for experiments, cells werepelleted by centrifugation at 2,000 g for 5 minutes. The supernatant wasdiscarded and the cell pellet was resuspended in phosphate bufferedsaline (PBS). The concentration of E. coli was re-adjusted to be 1.0×10⁹CFU/mL (OD₆₀₀=1.0).

To label the E. coli with the green fluorescent label SYTO 9™ (LifeTechnologies, Grand Island, N.Y.), which selectively labels livebacteria, the E. coli solution was incubated with 300× diluted SYTO 9for 30 minutes at room temperature, followed by centrifugation andredispersion in PBS. The labeled E. coli (1.0×10⁷ CFU/mL) andnanoparticles (1.0×10¹¹/mL) were mixed in PBS and incubated for oneminute at room temperature. This concentration of bacteria was selectedbased on the reported peak blood concentration of E. coli afterinjection of a lethal dose (LD100) in vivo. 10 μL of this mixture wasadded to a cell counting chamber for visualization by opticalmicroscopy.

An external permanent magnet was applied to the chamber and draggedacross its bottom surface, causing clusters of NP_(PEG-DPA-Zn)co-localized with E. coli to migrate up several centimeters. Phasecontrast and green fluorescent images were taken of the chamber usingOlympus FSX100 (Olympus, Japan) to check the co-localization of MNPs andE. coli.

Referring to FIGS. 10A-10C, the co-localization of NP_(PEG-DPA-Zn) andE. coli was evidenced by the overlap of a phase contrast image 150 (FIG.10A) showing NP_(PEG-DPA-Zn) and a green fluorescent image 152 (FIG.10B) showing E. coli. The co-localization can also be seen clearly in anoverlay image 154 (FIG. 10C) including both phase contrast andfluorescent image data. This overlap is indicative of the binding ofNP_(PEG-DPA-Zn) to E. coli. The scale bar in all images of FIG. 10denotes 100 μm.

Referring to FIGS. 10D-10F, a mixture of green fluorescence-labeled E.coli and NP_(PEG) was also prepared and the same magnetic experiment wasconducted. No co-localization of E. coli and NP_(PEG) was observed, ascan be seen from the lack of overlap between a phase contrast image 156(FIG. 10D) showing NP_(PEG) and a green fluorescence image 158 (FIG.10E) showing E. coli. An overlay image 160 (FIG. 10F) also shows thatthere is no significant overlap between E. coli and NP_(PEG). This lackof overlap indicates that there is no interaction between the PEG chainsand the E. coli, and also suggests that complete passivation of theamine groups on the surface of the nanoparticles has occurred.

The specificity of the binding between bis-Zn-DPA and E. coli wasfurther demonstrated by adding fluorescein (FITC)-labeled bis-Zn-DPA-PEGmolecules (bis-Zn-DPA-PEG-FITC) to unlabeled E. coli. Fluorescenceprobes (100 μM), including bis-Zn-DPA-PEG-FITC, FITC-labeledbis-DPA-PEG, FITC-labeled Zn—NH₂-PEG, and FITC-labeled NH₂-PEG, wereeach mixed with a sample of unlabeled E. coli (1.0×10⁷ CFU/mL). After 30minutes of incubation, each sample was washed twice with PBS and imagedunder optical and fluorescence microscopy.

After adding bis-Zn-DPA-PEG-FITC to E. coli, fluorescence microscopyshowed that most E. coli were stained with green fluorescence,confirming the binding of bis-Zn-DPA-PEG-FITC to E. coli. Minimalfluorescence was detected when FITC-labeled bis-DPA-PEG (without Zn²⁺)was added to E. coli, indicating that Zn participates in the specificbinding to E. coli. FITC-labeled PEG and FITC-labeled amine moleculesalso did not stain E. coli effectively regardless of the addition of Zn,indicating that binding of these molecules to E. coli was minimal.

Example 3 Separation of E. Coli from a Buffer Solution

The efficiency of modified nanoparticles to separate a fixedconcentration of E. coli (1.0×10⁷ CFU/mL) by magnetic separation wasmeasured in phosphate buffered saline (PBS) with varying concentrationsof NP_(PEG-DPA-Zn) DPA-Zn and NP_(PEG) (from 0 to 1.0×10¹¹/mL). Eachmixture of E. coli and nanoparticles was incubated in a 1.5 mL test tubefor one minute at room temperature and a permanent magnet was placed onthe side of the test tube for 2 minutes. 10 μL of the supernatant wascollected and added to a cell counting chamber for analysis.

The number of bacteria in a 100× green fluorescence image of thesupernatant was counted (using the NIH-provided freeware ImageJ) andconverted to E. coli concentration in the solution. For this, a standardcurve was generated that correlated the number of E. coli in the 100×green fluorescence image with the E. coli concentration in the solution(see below).

Referring to FIG. 11, a concentration of 1.4×10¹⁰/mL of NP_(PEG-DPA-Zn)removed 50% of E. coli and a concentration of 1.0×10¹¹/mL ofNP_(PEG-DPA-Zn) completely cleared E. coli from the solution (curve180). In contrast, NP_(PEG) did not remove E. coli even at the highestMNP concentration (curve 182). In FIG. 12, E. coli concentrations werenormalized to the E. coli concentration prior to separation. The datawere fitted with a sigmoid curve and a straight line with a zero slopefor curves 180 and 182, respectively. Data are medians with 25^(th) and75^(th) percentiles (N=4).

The enumeration of bacteria using fluorescent labeling and microscopicanalysis is a facile and reliable alternative to colony counting. Toidentify a correlation between the fluorescent and the colony-countingmethods, ten-fold serial dilutions (1:10, 1:100, 1:1,000, 1:10,000, and1:100,000) of SYTO 9-labeled E. coli in PBS (OD₆₀₀=1.0, 1.0×10⁹ CFU/mL)were used to count the number of bacteria in solution using fluorescentmicroscopy and bacterial culture on agar plates. For fluorescentcounting, 10 μL of each bacterial cell solution was added to a Countess™cell counting chamber slide (Life Technologies, Grand Island, N.Y.), andthe number of bacteria in a 40× green fluorescence image was countedusing ImageJ. For bacterial culture and subsequent colony counting, eachbacterial solution was further diluted and 200 μL of the dilutedbacterial solution was plated onto an LB-agar plate such that 200-300colony forming units grew per plate. Four plates were created for eachserially diluted bacterial cell solution. The two types of bacterialcell count data were plotted against each other on a logarithmic scaleto confirm linearity, as shown in a plot 250 of FIG. 12.

In this example and in other examples described herein, a normaldistribution of data was not assumed due to the asymmetric distributionof some of the data. Data were analyzed by the non-parametricMann-Whitney U-test. A p value<0.05 was considered statisticallysignificant unless stated otherwise. For multiple comparisons,Kruskal-Wallis tests were performed followed by Bonferroni corrections.

Example 4 Separation of Endotoxins from a Buffer Solution

To study the ability of NP_(PEG-DPA-Zn) to remove endotoxins, magneticseparation of endotoxins from solution was performed in a test tube withvarious concentrations of NP_(PEG-DPA-Zn) and NP_(PEG). Nanoparticles(with concentrations ranging from 4.0×10⁴ to 2.0×10⁶/mL) were added to astandard endotoxin solution in PBS of 1.0 EU/mL in 1:1 volume ratio(ToxinSensor™ Chromogenic LAL Endotoxin Assay Kit (Genscript USA Inc.,Piscataway, N.J.). After incubation for 1 minutes at room temperature ina test tube, an external magnetic field was applied, separating thenanoparticles from the solution. The remaining solution was collectedand endotoxins in the collected solution were measured by using theToxinSensor™ Chromogenic LAL Endotoxin Assay Kit.

Referring to FIG. 13, NP_(PEG-DPA-Zn) decreased the concentration offree endotoxin in PBS as the concentration of NP_(PEG-DPA-Zn) increased,to as low as 9.0% of the starting concentration (curve 350). NP_(PEG)did not affect the free endotoxin concentration at any nanoparticleconcentration (curve 352). These results demonstrate thatNP_(PEG-DPA-Zn) can separate endotoxins from solution throughmagnetophoresis. In FIG. 14, endotoxin concentrations were normalized tothe endotoxin concentration prior to separation. The data were fittedwith a sigmoid curve and a straight line with a zero slope for curves350 and 352, respectively. Data are medians with 25th and 75thpercentiles.

Example 5 Separation of E. Coli from Blood in a Microfluidic System

The feasibility of using NP_(PEG-DPA-Zn) to remove bacteria from bloodwas tested for bovine blood (Bovine whole blood with heparin (0.5 U/mL),Sierra for Medical Science, Whittier, Calif.) diluted to a red bloodcell (RBC) concentration of 1.0×10⁸/mL. This concentration,approximately 50 times lower than that of adult human blood(˜5.0×10⁹/mL), was used because higher RBC concentrations obstructedmicroscopic imaging after magnetic separation. Any changes in the RBCconcentration after magnetic separation indicate the degree ofnon-specific binding between nanoparticles and RBCs.

Nanoparticles (1.0×10¹¹/mL) and E. coli (1.0×10⁷ CFU/mL) were mixed withbovine RBC solution (1.0×10⁸/mL) and incubated for one minute at roomtemperature. After magnetic separation in a test tube as describedabove, green fluorescence and phase contrast images were used to countthe number of E. coli and RBCs, respectively. After the first round ofseparation was performed, fresh nanoparticles were added to thesolution, and another round of magnetic separation was performed. Thisprocess was repeated until no E. coli were found in the solution.

Referring to FIG. 14A, NP_(PEG-DPA-Zn) (curve 450) removed about 70% ofE. coli from the blood sample during a first cycle, a 30% decrease inthe amount of removed E. coli compared to the same separation processconducted in PBS (FIG. 11). A second round of magnetic separation on thesame blood sample with fresh NP_(PEG-DPA-Zn) resulted in completeremoval of E. coli from the blood sample. Referring to FIG. 14B, thenumber of RBCs did not change during these separations (curve 454),indicating negligible non-specific interactions between NP_(PEG-DPA-Zn)and RBCs. Similar experiments with NP_(PEG) did not alter the numbers ofE. coli (curve 452) or RBCs (curve 456) after either the first or thesecond separation, indicating that NP_(PEG) are incapable of selectivelyseparating E. coli and that NP_(PEG) do not bind non-specifically toRBCs. In FIGS. 14A and 14B, RBC concentrations were normalized to theirconcentrations prior to separation. Data are medians with 25th and 75thpercentiles. * indicates p<0.05 compared to the initial concentrations.

A hemolysis assay confirmed that minimal damage was done to RBCs byNP_(PEG-DPA-Zn) or NP_(PEG). To evaluate the degree hemolysis induced bythe presence nanoparticles, RBCs were collected from bovine whole bloodby centrifugation at 2,000 g for 15 minutes. Saline was added to theconcentrated RBC solution to recover the original RBC concentration.Nanoparticles (1.0×10¹¹/mL of NP_(PEG-DPA-Zn) and NP_(PEG)) were addedto the RBC solution. As a negative control, in which RBCs were known notbe harmed, saline was added to the RBC solution instead ofnanoparticles. As a positive control, in which all RBCs are lysed,Triton X-100 (1.0% v/v) was added to the RBC solution. After incubationat 4° C. for 30 minutes, the RBC mixtures were centrifuged at 15,000 gfor 5 minutes and the absorbance (Abs) of the supernatant at 541 nm (anabsorbance peak of hemoglobin) was measured.

The percentage of hemolysis was calculated by the following equation:

${\% \mspace{14mu} {hemolysis}} = {\frac{{{Abs}\mspace{14mu} {of}\mspace{14mu} {sample}} - {{Abs}\mspace{14mu} {of}\mspace{14mu} {negative}\mspace{14mu} {control}}}{{{Abs}\mspace{14mu} {of}\mspace{14mu} {positive}\mspace{14mu} {control}} - {{Abs}\mspace{14mu} {of}\mspace{14mu} {negative}\mspace{14mu} {control}}} \times 100}$

Referring to Tables 2 and 3, neither NPPEG nor NP_(PEG-DPA-Zn) causedany significant hemolysis. Data in Table 2 are the mean absorbance at541 nm with standard deviations of the supernatants from the hemolysisassay described above. Data in Table 3 are means with standarddeviations. All samples were diluted ten times before measurement (N=4).

TABLE 2 Absorbance at 541 nm from exposure to nanoparticles, withcontrols Agent Triton X-100 Saline NPPEG NP_(PEG-DPA-Zn) Abs at 541 nm0.837 ± 0.052 0.093 ± 0.003 0.915 ± 0.002 0.09475 ± 0.002

TABLE 3 Percentage of hemolysis from exposure to nanoparticlescalculated using the equation above Agent NP_(PEG) NP_(PEG-DPA-Zn) %Hemolysis −0.23 ± 0.35 0.20 ± .022

Example 6 Separation of E. Coli in a Network of Microfluidic Systems

A microfluidic system (1,000 μm width×200 μm height) was fabricated bycasting poly(dimethylsiloxane) (PDMS, Dow Corning, Midland, Mich.) ontoa silicon substrate with channels patterned with SU-8 (Microchem,Newton, Mass.). After curing the PDMS at 65° C. for 2 hours, inlets andoutlets for fluids were created using a 1.0 mm biopsy punch. To hold thedisk magnets (12.5 mm diameter×5.0 mm height, NdFeB, 3.4×10−3 G (gauss)in 1.0 mm, Indigo Instruments, Canada) in position, three 12-mm holeswere created 2.5 mm from the channel, in series along the direction offlow in the microchannel. The PDMS slab with the pattern ofmicrochannels was subsequently sealed onto glass (Dow Corning).

Referring again to FIG. 5, in the dual inlet microfluidic system 600,saline (9.0% NaCl, w/v) was injected into inlet A. A mixture of RBCs(1.0×10⁸ RBC/mL), fluorescence-labeled E. coli (1.0×10⁷ CFU/mL), andNP_(PEG-DPA-Zn) or NP_(PEG) nanoparticles (1.0×10¹¹ particles/mL) wasinjected into inlet B. Three permanent magnets 610 were placed along themain channel to effect magnetophoric separation of E. coli from thesaline. Liquid exited the system through outlets C and D. A flow rate of9.0 mL/hour was used.

Referring to FIGS. 15A-15E, the migration of RBCs and labeled E. coliwas visualized by phase contrast and fluorescence microscopy for avariety of flow conditions. In these images, the clear layer of flow wassaline and the turbid layer contained nanoparticles, RBCs, and E. coli.Nanoparticles appear dark in phase contrast images; E. coli appearbright in fluorescence images. The top and bottom flow channel branchesat the right of each image point to outlets C and D, respectively, ofthe microfluidic system.

Images 550 a and 550 b (FIG. 15A) correspond to the flow of a solutionincluding NP_(PEG-DPA-Zn) with no magnet present along the channel.Without a magnetic field, nanoparticles, RBCs, and E. coli exited thechannel through outlet D.

Images 552 a and 552 b (FIG. 15B) and images 554 a and 554 b (FIG. 15C)correspond to the flow of a solution including with the magnet presentalong the channel. With a magnetic field, E. coli that had bound toNP_(PEG-DPA-Zn) were pulled toward the magnet and exited through outletC. A significant portion of the E. coli and nanoparticles also clusteredaround the magnet (FIG. 15C) rather than exiting through outlet C.

Images 556 a and 556 b (FIG. 15D) correspond to the flow of a solutionincluding NP_(PEG) for the control situation in which no magnet waspresent along the channel. Images 558 a and 558 b (FIG. 15E) correspondto the flow of a solution including NP_(PEG) with the magnet presentalong the channel. The bulk of the turbid layer of flow was unaffectedby the magnetic field and exited through outlet D. E. coli did notmigrate toward outlet C nor did E. coli cluster around the magnet.

Referring to FIG. 16, the E. coli concentration was measured at outlet Cfor flows including NP_(PEG-DPA-Zn) and NP_(PEG). About 25% of theoriginal concentration of E. coli was measured at outlet C after asingle pass of the mixture through the dual inlet system in the presenceof the magnet (bar 650); no E. coli were observed with no magnet (bar652). No E. coli were observed with (bar 654) or without (Bar 656) themagnet for mixtures containing NP_(PEG). Data are medians with 25th and75th percentiles. * indicates p<0.05 compared to NP_(PEG) with nomagnet.

The dual inlet system was also used for separation of E. coli from wholeblood. Although more E. coli were found to be associated withNP_(PEG-DPA-Zn) than with NP_(PEG) at outlet C when using whole blood,many RBCs, E. coli, and nanoparticles also exited through outlet C evenin the absence of a magnetic field. This result may be due to thedifficulties in maintaining two symmetric laminar flows when using wholeblood, which has a larger viscosity than saline (viscosity=1 cP forsaline, 10 cP for blood).

Referring again to FIG. 6, a single inlet, dual outlet microfluidicsystem 600 was prepared by closing the inlet A. Nanoparticles(1.0×10¹¹/mL) and fluorescence-labeled E. coli (5.0×10⁶ CFU/mL) weremixed with whole blood and injected into inlet B at 9.0 mL/hour. Threepermanent magnets were placed along the main channel to effectmagnetophoric separation of E. coli from whole blood. Liquid exited thesystem through outlets C and D and the liquid exiting through outlet Dwas collected and analyzed with fluorescent microscopy. All experimentswere performed at 4° C. to prevent blood coagulation.

Referring to FIG. 17A-17F, the colocalization of NP_(PEG-DPA-Zn) (FIGS.17A and 17B) or NP_(PEG) (FIGS. 17D and 17E) with fluorescence-labeledE. coli within the main channel of the microfluidic system wasvisualized by phase contrast and fluorescence microscopy, respectively.Overlay images show the overlap between the corresponding phase contrastand fluorescence images for NP_(PEG-DPA-Zn) (FIG. 17C) and NP_(PEG)(FIG. 17F). These images demonstrate that NP_(PEG-DPA-Zn) are co-locatedwith E. coli near the magnets, while NP_(PEG) accumulated near themagnets without E. coli.

Referring to FIG. 18, after each pass through the single inletmicrofluidic system, the solution was collected at outlet D andre-infused at inlet B with a fresh infusion of nanoparticles. Thisprocess was repeated four times. For solutions containingNP_(PEG-DPA-Zn), all E. coli had been separated from whole blood withintwo cycles of separation through the microfluidic system (curve 850).For solutions containing NP_(PEG), no change in the E. coli populationwas observed (curve 852). In FIG. 18, the concentration of E. coli inwhole blood at outlet C was normalized to the initial E. coliconcentration. Data are medians with 25th and 75th percentiles (N=4). *indicates p<0.01 compared to the initial concentration.

The effectiveness of microfluidic separation was enhanced by flowingblood through a nested microfluidic system formed by connecting multipleindividual microfluidic systems in series and in parallel. In the nestedmicrofluidic system, multiple magnetic separations could be completed ina single pass. Referring again to FIG. 7, the nested system 700 had asingle inlet B, through which a mixture of E. coli (5.0×106 CFU/mL),nanoparticles (1.0×1011/mL), and whole blood was injected. Blood exitingthe system 700 at various outlets was collected and the concentration ofE. coli in each sample was determined. Blood exiting through outlets Gand G had passed through two magnetic separation sites; blood exitingthrough outlets I and J had passed through three magnetic separationsites. Various flow rates (9, 18, 30, and 60 mL/hour) of blood throughthe system were tested.

Referring to FIGS. 19A-19D, the concentration of E. coli at sites G, H,I, and J was determined for each flow rate and is shown in graphs 950,952, 954, 956, respectively. For solutions containing NP_(PEG-DPA-Zn),the concentration of E. coli in blood exiting through outlets G and Hwas about 20% of the starting concentration for all flow rates. Muchlower concentrations (<5%) were found in blood exiting through outlets Iand J. These significant reductions in bacterial concentration werereproduced for flow rates as high as 60 mL/hour. There was nostatistically significant difference between concentrations at G and Hor between concentrations at I and J. There was also no statisticallysignificant difference in concentrations for the four flow rates.

For solutions containing NP_(PEG) , E. coli concentrations remainedsubstantially the same as the initial E. coli concentration regardlessof the position of the outlet or the flow rate. In FIGS. 19A-19D, theconcentration of E. coli in whole blood at each outlet was normalized tothe initial E. coli concentration. Data are medians with 25th and 75thpercentiles (N>5). * indicates p<0.05 compared to the initialconcentration.

Example 7 Protocol for In Vivo Separation

To demonstrate that bacterial separation can work for in vivo systems,an animal model protocol was developed. PDMS “chips” including multipleparallel microfluidic channels were prepared. The microfluidic channeldesign emulated capillary microcirculation, which maintains theviability of fragile circulating red and white blood cells as well aspreventing thrombosis (clot). The channel diameter was determined basedon a balance between having sufficiently large channel diameters tominimize shear stress and flow irregularities and the loss of functionalsurface area that results from increasing diameter.

To demonstrate that bacterial separation/filtration system andextracorporeal circuitry works in vivo, an established rat model ofseptic rats placed onto cardiopulmonary bypass circuits can be used.Adult male Sprague-Dawley rats can be rendered septic by cecal ligationand puncture (i.e. their bowel will be punctured to induce septicshock), mechanically ventilated, and then cannulated at the femoral vein(to drain blood) and jugular vein (to return blood). By this paradigm,blood is removed from the body, pumped through bacteria separationsystem, and returned to the body. Bacterial cells can be quantifiedbefore and after treatment by counting the number of colonies forming onbacterial plate cultures. Further, concentration of endotoxin in theblood can be determined using commercially available assays.

Example 8 Ligand Synthesis

FIG. 20 shows a scheme 10 for the synthesis of3-(3,5-bis((bis(pyridin-2-ylmethyl)amino)methyl)-4-hydroxyphenyl)propanoic acid (bis-DPA). Paraformaldehyde (PFA, 0.55 g, 18 mmol) anddi(2-picolyl)amine (DPA, 2.8 g, 14 mmol) were placed in a 250 mL roundbottom flask and suspended in a mixture of ethanol (15 mL) and water (45mL). Commercially available methyl 3-(4-hydroxyphenyl)propanoate (MHPP,1.0 g, 5.6 mmol) and hydrochloric acid (HCl, 1.0 M, 1.4 mL) were addedto the mixture and refluxed for 2 days. After the reaction mixture wascooled to room temperature, it was neutralized with saturated Na₂CO₃ andthe aqueous solution was extracted with excess chloroform. An organicphase was dried over Na₂SO₄ and chloroform was evaporated under reducedpressure. The crude product was purified by column chromatography onsilica gel (MeOH:CHCl₃ 5:95) to obtain methyl3-(3,5-bis((bis(pyridin-2-ylmethyl)amino)methyl)-4-hydroxyphenyl)propan-oate,bis-DPA-methyl ester (bis-DPA-ME) as a pale yellow oil (2.4 g, 4.0 mmol,71%).

A solution of bis-DPA-ME (2.4 g, 4.0 mmol) in tetrahydrofuran (THF, 330mL) and methanol (210 mL) was added to a LiOH solution (aq., 2.0 M, 200mL). The mixture was stirred for 12 hours at room temperature and thenpoured into chloroform (300 mL). After acidification with HCl (1.0 M),the aqueous layer was extracted with excess chloroform and the organicphase was dried over Na₂SO₄. Bis-DPA was obtained without furtherpurification after complete evaporation of chloroform.

The synthesis of bis-DPA was confirmed by ¹H nuclear magnetic resonance(NMR) spectroscopy in DMSO-d₆ (400 MHz, Varian), as shown in the NMRspectrum S200 of FIG. S2. The peaks were identified as follows: δ 10.89(bs, 1H), 8.48 (d, 4H), 7.68 (t, 4H), 7.46 (t, 4H), 7.21 (d, 4H), 7.04(s, 2H), 3.70 (s, 8H), 3.57 (s, 4H), 2.62 (t, 2H), 2.46 (t, 2H).

FIG. 21 shows a scheme 20 for the synthesis of bis-DPA-PEG-COOH (10kDa). Bis-DPA (0.1 g, 0.17 mmol),1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC, 65mg, 0.34 mmol) and N-hydroxysuccinimide (NHS, 23 mg, 0.2 mmol) wereadded to and stirred in dry dichloromethane (10 mL) for 4 hours at roomtemperature. The solvent was removed under reduced pressure and thecrude product was purified by flash chromatography (MeOH:CHCl₃ 5:95) toobtain DPA-succinimidyl valerate (DPA-SVA, 76 mg, 0.11 mmol, 65%) as apale yellow solid. A mixture of NH₂-PEG-COOH (10 kDa, 0.92 g, 0.092mmol, JenKem Technology USA, Allen, Tex.) and DPA-SVA (76 mg, 0.11 mmol)was stirred for 2 hours in dry dichloromethane withN,N-diisopropylethylamine (DIPEA, 32 μL). bis-DPA-PEG-COOH wasquantitatively obtained after size exclusion chromatography inchloroform.

The synthesis of bis-DPA-PEG-COOH was confirmed by ¹H NMR spectroscopyin DMSO-d₆ (400 MHz, Varian), as shown in the NMR spectrum S400 of FIG.S4. The peaks were identified as follows: δ 8.43 (d, 4H), 7.93 (bs, 1H),7.68-7.64 (m, 4H), 7.41 (d, 4H), 7.19-7.16 (m, 4H), 6.96 (s, 2H), 3.94(s, 8H), 3.63 (m, 8H), 3.44 (s, 960), 3.27 (m, 2H), 2.91 (t, 2H), 2.45(m, 4H).

FIG. 22 shows a scheme 30 for the synthesis of bis-Zn-DPA-PEG-FITC (10kDa). A mixture of NH₂-PEGFluorescein isothiocyanate (FITC, 10 kDa, 20mg, 2.0 μmol) and DPA-SVA (1.6 mg, 2.4 μmol) was stirred for 2 hours indry dichloromethane with N,N-Diisopropylethylamine (DIPEA, 1.0 μL).bis-DPA-PEG-FITC was quantitatively obtained after size exclusionchromatography in chloroform. bis-DPA-PEG-FITC (20 mg, 2.0 μmol) andzinc nitrate hexahydrate (0.76 mg, 4.0 μmol) were dissolved in methanol(2.0 mL). The mixture was stirred for 1 h at room temperature and thesolvent was evaporated under reduced pressure to obtainbis-Zn-DPA-PEG-FITC.

Cu-DPA-Cl₂ was synthesized by adding DPA (104 mg, 0.5 mmol) to asolution of CuCl₂.2H₂O (82 mg, 0.5 mmol) in methanol (20 mL). Themixture was refluxed for one hour, allowed to cool to room temperature,and filtered. The filtration product was allowed to crystallize at roomtemperature and then filtered and recrystallized from a hotwater/acetonitrile (1:1, 10 mL) mixture. From the Cu-DPA-Cl₂ product,Cu-DPA-PEG-COOH or Cu-DPA-PEG-FITC can be obtained using standardchemical reactions. Further details about the synthesis andcharacterization of Cu-DPA-Cl₂ can be found in Choi et al., “Synthesis,properties, and X-ray structure of [Cu(dpa)Cl₂](dpa=di-(2-picolyl)amine),” Journal of Chemical Crystallography,December 2003, 33:12, pp 947-950, the contents of which are incorporatedherein by reference in their entirety.

Unless otherwise specified, chemicals were purchased from Sigma-Aldrich(St. Louis, Mo.) and used without further purification.

OTHER EMBODIMENTS

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A nanoparticle configured to bind to a bacterialcontaminant, the nanoparticle comprising: a core comprising a magneticmaterial; and a plurality of ligands attached to the core, wherein theligands comprise bis(dipicolylamine) coordinated with a zinc ion(bis-Zn²⁺-DPA), wherein the bis-Zn²⁺-DPA can bind to the bacterialcontaminant.
 2. The nanoparticle of claim 1, wherein the bis-M-DPA isattached to the core via a spacer molecule having a molecular weight of10 kDa to 50 kDa.
 3. The nanoparticle of claim 2, wherein the spacer isa polymer spacer.
 4. The nanoparticle of claim 1, wherein the ligandsfurther comprise a filler molecule attached to the core.
 5. Thenanoparticle of claim 1, wherein the core comprises one or more of aferrimagnetic material, a ferromagnetic material, an antiferromagneticmaterial, a paramagnetic material, and a superparamagnetic material. 6.The nanoparticle of claim 1, wherein the core comprises iron oxide. 7.The nanoparticle of claim 1, wherein an external surface of the corecomprises a chemical group that can bind to the ligands.
 8. Thenanoparticle of claim 1, wherein the nanoparticle is configured suchthat binding between the nanoparticle and the bacterial contaminant issubstantially complete within an incubation time of about five minutesor less.
 9. The nanoparticle of claim 1, wherein the bacterialcontaminant includes at least one of Gram positive bacteria, Gramnegative bacteria, and an endotoxin.
 10. A method of separating abacterial contaminant from a liquid, the method comprising: combiningthe liquid with a plurality of nanoparticles that can bind to thebacterial contaminant, each nanoparticle comprising: a core comprising amagnetic material, and a plurality of ligands attached to the core, theligands comprising bis(dipicolylamine) coordinated with a zinc ion(bis-Zn²⁺-DPA); applying a magnetic field to the liquid including thenanoparticles, wherein at least some of the nanoparticles are attractedby the magnetic field; and separating the liquid from the attractednanoparticles, wherein the bacterial contaminant, if present in theliquid, is bound to at least some of the attracted nanoparticles. 11.The method of claim 10, wherein the liquid includes blood.
 12. Themethod of claim 10, further comprising directing the liquid includingthe nanoparticles through a flow channel.
 13. The method of claim 12,wherein directing the liquid through the flow channel includes flowingthe liquid through the flow channel with a flow rate of at least about 9mL/hour.
 14. The method of claim 13, further comprising incubating theliquid including the nanoparticles prior to applying the magnetic fieldfor about five minutes or less to achieve substantially complete bindingbetween the nanoparticles and the bacterial contaminant.
 15. A systemfor separating a bacterial contaminant from a liquid, the systemcomprising: a plurality of nanoparticles that can bind to the targetspecies, each nanoparticle comprising: a core comprising a magneticmaterial, and a plurality of ligands attached to the core, the ligandscomprising bis(dipicolylamine) coordinated with a zinc ion(bis-Zn²⁺-DPA); a source of a magnetic field arranged to apply themagnetic field to a liquid including the plurality of nanoparticles,wherein at least some of the nanoparticles are attracted by the magneticfield; and a flow path for separating the liquid from the attractednanoparticles, wherein the bacterial contaminant, if present in theliquid, is bound to at least some of the attracted nanoparticles. 16.The system of claim 15, further comprising one or more flow channelsconfigured to receive the liquid including the nanoparticles.
 17. Thesystem of claim 16, wherein the source of the magnetic field is disposedto a side of the one or more flow channels and configured to apply themagnetic field across the one or more flow channels.